Hepatitis B virus (HBV) is a virus with which approximately 240 million people are chronically infected to chronically affect chronic HBV carriers, causing public health problems all over the world. An HBV envelope includes three associated surface glycoproteins, particularly a large (L) protein, a middle (M) protein, and a small (S) protein. These proteins are products which originate from one open reading frame (ORF), and are classified into pre-S1, pre-S2 and S domains. The S protein is encoded in the S domain, and the M protein includes pre-S and S antigens. Also, the L protein includes pre-S1, pre-S2 and S antigens. The three antigens are known to stimulate the production of virus neutralizing antibodies. In particular, the ‘a’ determinants of the pre-S1 and S antigens against the L protein were reported to play a critical role in virus infection. More specifically, the HBV receptor of liver cells has been identified in recent years. It was found that a protein referred to as a sodium taurocholate transporter (NTCP) is a high-affinity functional receptor that binds to HBV pre-S1 (Yan, H., et al., Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. Elife, 2012. 1: p. e00049). Also, it was found that heparin sulfate proteoglycan (HSPG) is a low-affinity receptor that binds to the a determinant of the S antigen (Sureau, C. and J. Salisse, A conformational heparan sulfate binding site essential to infectivity overlaps with the conserved hepatitis B virus a-determinant. Hepatology, 2013. 57(3): p. 985-94). A region of pre-S1 binding to the NTCP consists of amino acid residues at positions 2 to 47 of an ayw subtype, and consists of amino acid residues at positions 13 to 58 of an adr subtype. These results suggest that the antibody binding to a receptor-binding site of pre-S1 can very effectively serve to inhibit the infection of HBV.
For the immunoprophylaxis of HBV infection, hepatitis B immune globulin (HBIG) prepared from the human anti-HBsAg plasma is administered to infants born from HBsAg-HBeAg-positive parents, susceptible subjects acutely exposed to infectious HBV-containing materials, liver transplant patients having chronic HBV-associated liver diseases, etc. However, HBIG cannot be considered to be an ideal source for antibodies since it exhibits limited obtainability and poor specific activity. In this circumstance, the virus-neutralizing monoclonal antibody against the pre-S1 and S antigens may be an effective substitute for the immunoprophylaxis of HBV infection. Based on this situation, the present inventors have developed a humanized antibody through the grafting of murine monoclonal antibodies KR127 and CDR that recognize amino acid residues (NSNNPDWDF) (this sequence corresponds to 26th to 34th amino acid residues of the ayw subtype) of HBV pre-S1 of the adr subtype (Korean Registered Patent No. 10-0345463). However, since the humanized antibody has a relatively low antigen-binding affinity and includes a considerable number of amino acid residues derived from a mouse, there has been a lasting demand for development of antibodies that have an effect of inducing an effective neutralization reaction to pre-S1 antigens and simultaneously exhibit lower immunogenicity in humans.
In the last 30 years, monoclonal antibodies (mAbs) have emerged as a potent therapeutic agent in humans with advances in antibody engineering technology. Since non-human antibodies may stimulate an immune response in humans, their therapeutic use has been restricted. To solve these problems, the humanized antibodies have been prepared using the CDR grafting technology of engrafting a complementarity-determining region (CDR) of a murine antibody into a human framework region (FR). However, since some FR residues come in direct contact with antigens or serve to hold a CDR loop structure, the simple CDR grafting often reduces affinity. Therefore, the humanized antibody should be usually prepared using methods other than the CDR grafting to preserve some murine FR residues. However, such a humanized antibody generally induces an immune response in humans at a lower level, compared to the chimeric antibodies, but still exhibits immunogenicity since the CDR is not derived from a human being. Accordingly, there is a demand for development of antibodies capable of minimizing immunogenicity of the humanized antibody while maintaining the affinity to antigens.